1) Field of the Invention
The present invention relates to a method for the determination of the presence of a reverse transcriptase in a sample liquid and also to a method for the determination of infection of a patient to a retrovirus on the basis of measurement of the reverse-transcriptase-neutralizing and/or inhibiting antibody titer in a sample body fluid.
2) Description of the Related Art
For the measurement of the activity of a reverse transcriptase, there has been reported a method in which a native RNA or adeninc ribopolynucleotide (which may hereinafter be abbreviated as "poly A") and an oligodeoxythyminenucleotide (which may hereinafter be abbreviated as "oligo dT") and as a substrate, a tritium labeled deoxythymidine 5'-triphosphate (which may hereinafter be abbreviated as "[.sup.3 H]dTTP") are used, the oligo dT is allowed to elongate using the activity of the reverse transcriptase in a reaction mixture, the reaction mixture is filtered by a filter, and the residual radioactivity on the filter is then measured [David Baltimore, Nature, 226, 1209-1211 (1970); Howard M. Temin & Satoshi Mizutani, Nature, 226, 1211-1213 (1970)].
There has also been reported a method for the diagnosis of infection to an AIDS virus, a sort of retrovirus, by making use of the above-mentioned measurement method of the activity of a reverse transcriptase [Japanese Patent Application Laid-Open (Kokai) No. 252253/1988].
Many of the procedures which have heretofore been used commonly in biopharmaceutical research and recombinant DNA technology greatly rely upon the use of polynucleotides generally radiorabelled by isotopic hydrogen (.sup.3 H), phosphorus (.sup.32 P), carbon (.sup.14 C) or iodine (.sup.126 I). Such radioactive elements are employed as useful indicator probes because they can detect, monitor, localize or isolate nucleic acids or other scientifically or clinically interesting molecules even when such nucleic acids or molecules are present in a trace amount.
Stringent limitations are however imposed on the use of radioactive substances, resulting in serious drawbacks. As a first problem, for those handling a radioactive substance, there is a potential danger that they can be exposed to high-level radioactive rays. Careful and fail-free safety measures must be practiced during the production, use and post treatment of radioactive isotopes. A second problem resides in that a radioactive nucleotide is expensive and its use results in a higher cost in general applications. As a third problem, a radioactive substance of highly specific radioactivity must be used to obtain a necessary level of sensitivity. The radioactive substance of high specific radioactivity however has a short half-life correspondingly, so that its shelf life is limited. This imposes limitations on its use.
It has hence been studied to use a nonradioactive labeling substance in place of such a radioactive substance. It has however been reported, for example, that when a native mRNA as a template, oligo dT as a primer and a biotinylated deoxyuridine 5'-triphosphate (which may hereinafter be abbreviated as "biotin-dUTP") as a substrate were reacted using a reverse transcriptase derived from avian myeloblastosis virus, biotin-dUTP was not used at all as a substrate, no elongation of oligo dT was observed, and biotin-dUTP cannot therefore be used for the measurement of the activity of the reverse transcriptase [Pennina R. Langer, Alex A. Waldrop & David C. Ward, Proc. Natl. Acad. Sci. USA, 78, 6633-6637 (1981)]. Biotinylated nucleotides have therefore been considered to be unapplicable for the measurement of the activities of reverse transcriptases.
The present inventors previously conducted an extensive investigation with a view toward obtaining a measurement system for the activity of a reverse transcriptase without using any radioactive isotope. As a result, it was found that the use of a specific RNA template and a particular primer makes it possible to use biotin-dUTP as a substrate in the presence of a reverse transcriptase and the activity of the reverse transcriptase can thus be easily measured.
The above method, however, still involves some problems so that there has been a demand for their improvements. Described specifically, the above method is accompanied by the problems that it requires cumbersome procedures and some time is needed until measurement becomes feasible after initiation of the reaction. It has therefore been desired to develop a method which is simple in procedures and permits measurement in a short time.